Kerbs Anastasia, Mindt Melanie, Schwardmann Lynn, Wendisch Volker F
Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University, 33615 Bielefeld, Germany.
BU Bioscience, Wagenigen University and Research, 6700AA Wageningen, The Netherlands.
Microorganisms. 2021 Apr 13;9(4):824. doi: 10.3390/microorganisms9040824.
alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to alkylated amino acids like methyl-l-alanine or methylantranilate, a precursor of acridone alkaloids, have been established using engineered , which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of for de novo production of methylphenylalanine based on reductive methylamination of phenylpyruvate. Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of l-tryptophan and l-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of , methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an methylphenylalanine titer of 0.73 ± 0.05 g L, a volumetric productivity of 0.01 g L h and a yield of 0.052 g g glucose were reached. When xylose isomerase gene from and the endogenous xylulokinase gene were expressed in addition, xylose as sole carbon source supported production of methylphenylalanine to a titer of 0.6 ± 0.04 g L with a volumetric productivity of 0.008 g L h and a yield of 0.05 g g xylose. Thus, a fermentative route to sustainable production of methylphenylalanine by recombinant has been established.
烷基化氨基酸在自然界中广泛存在，也能在生物活性次生代谢产物中找到，如糖肽抗生素万古霉素和免疫抑制剂环孢素A。为满足对烷基化氨基酸的需求，目前它们是通过化学方法生产的；然而，这些方法往往缺乏对映体纯度，产品收率低，且需要有毒试剂。利用工程化的[具体微生物名称未给出]已经建立了生产烷基化氨基酸（如甲基-L-丙氨酸或吖啶酮生物碱的前体甲基邻氨基苯甲酸）的发酵途径，该[具体微生物名称未给出]已经用于氨基酸的工业生产数十年了。在此，我们描述了基于苯丙酮酸还原甲基化从头生产甲基苯丙氨酸的[具体微生物名称未给出]的代谢工程。含有氨基酸替换P262A和M141L的Δ-1-哌啶-2-羧酸还原酶DpkA对苯丙酮酸和丙酮酸表现出相当的催化效率，而野生型酶更倾向于后者底物而非前者。在工程改造以过量生产邻氨基苯甲酸的菌株中，缺失邻氨基苯甲酸合酶基因[具体基因名称未给出]以及编码支链氨基酸转氨酶IlvE和苯丙氨酸转氨酶AroT的基因，消除了L-色氨酸和L-苯丙氨酸的生物合成，从而积累苯丙酮酸。在[具体微生物名称未给出]异源表达后，向培养基中添加一甲胺可产生甲基苯丙氨酸。在基于葡萄糖的基本培养基中，甲基苯丙氨酸的滴度达到0.73±0.05 g/L，体积产率为0.01 g/(L·h)，葡萄糖产率为0.052 g/g葡萄糖。当另外表达来自[具体微生物名称未给出]的木糖异构酶基因[具体基因名称未给出]和内源性木酮糖激酶基因[具体基因名称未给出]时，以木糖作为唯一碳源可支持甲基苯丙氨酸的生产，滴度为0.6±0.04 g/L，体积产率为0.008 g/(L·h)，木糖产率为0.05 g/g木糖。因此，已经建立了通过重组[具体微生物名称未给出]可持续生产甲基苯丙氨酸的发酵途径。
